Newly Proliferated Cells in the Adult Male Amygdala
Are Affected by Gonadal Steroid Hormones
Christie D. Fowler,1 Marc E. Freeman,2 Zuoxin Wang1
1
Department of Psychology, Program in Neuroscience, Florida State University, Tallahassee, Florida
32306
2
Department of Biological Sciences, Program in Neuroscience, Florida State University, Tallahassee,
Florida 32306
Received 9 December 2002; accepted 19 May 2003
ABSTRACT:
Gonadal steroid hormones play an
important role in the proliferation, survival, and activation of neurons. The present study was performed to
examine the effects of testosterone and its metabolites on
newly proliferated cells in the amygdala of adult male
meadow voles (Microtus pennsylvanicus). Treatment
with testosterone propionate (TP) in castrated males
resulted in plasma testosterone levels similar to males
following mating. TP-treated males displayed a significant increase in the density of cells labeled with a cell
proliferation marker (BrdU) in the amygdala. Treatment with estradiol benzoate (EB) exerted a similar
effect as TP on the density of BrdU-labeled cells,
whereas 5␣-dihydrotestosterone (DHT) was ineffective.
A larger proportion (⬇44%) of the BrdU-labeled cells in
the amygdala displayed a neuronal phenotype, and a
INTRODUCTION
Adult mammalian neurogenesis may be influenced by
a variety of exogenous and endogenous factors. In the
dentate gyrus of the hippocampus (DG) of rodents,
the number of new cells increases in response to
environmental complexity (Kempermann et al., 1997;
Correspondence to: C. D. Fowler ([email protected]).
Contract grant sponsor: NIH; contract grant numbers: MH64352 and NS-07437 (C.D.F.), MH-58616 and MH-66734
(Z.X.W.), and DK-43200 (M.E.F.).
© 2003 Wiley Periodicals, Inc.
DOI 10.1002/neu.10273
lesser percentage (⬇35%) displayed a glial progenitor
phenotype; however, treatment effects were not found in
either population of cells. Hormonal effects appeared to
be site-specific as no group differences were found in the
dentate gyrus of the hippocampus or ventromedial hypothalamus. Finally, a time course study indicated that
BrdU-labeled cells in the amygdala are present as early
as 30 min following an acute injection of BrdU. Together, these data suggest that gonadal steroid hormones
influence the number of newly proliferated cells in the
amygdala, most likely by acting through an estrogenic
mechanism, and these effects may be exerted on locally
proliferating progenitors within the amygdala. © 2003
Wiley Periodicals, Inc. J Neurobiol 57: 257–269, 2003
Keywords: neurogenesis; testosterone; estrogen; DHT;
meadow vole
Nilsson et al., 1999), hippocampal-dependent learning
(Gould et al., 1999a), or wheel running (van Praag et
al., 1999). In contrast, psychosocial stress leads to a
decrease in the number of new cells in the DG of tree
shrews (Gould et al., 1997). The mechanisms underlying these effects may be attributed to changes in the
animal’s hormonal and/or neurotransmitter levels. For
example, the inhibitory effects of stress on neurogenesis may occur via adrenal steroids, such as glucocorticoids (Cameron and Gould, 1994; Fuchs et al.,
2001), whereas the enhancement of new cells may be
influenced by the presence of serotonin or brainderived neurotrophic factor (BDNF) (Brezun and
Daszuta, 1999, 2000; Pencea et al., 2001).
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Although cells undergoing neurogenesis in the
adult mammalian brain have been primarily documented in the DG and subventricular zone (SVZ),
several other brain regions have been identified to
contain newly proliferated cells. These regions include the amygdala and hypothalamus of voles and
hamsters (Huang et al., 1998; Fowler et al., 2002),
neocortex and amygdala of primates (Gould et al.,
1999b; Bedard et al., 2001), and striatum, septum, and
thalamus of rats (Pencea et al., 2001). Because the
amygdala is responsive to gonadal hormones (Insel,
1990; Coolen and Wood, 1999) and has been implicated in reproductive-associated behaviors, such as
olfactory/pheromonal processing (Luiten et al., 1985;
Meredith, 1991), social learning and memory (Kirkpatrick et al., 1994; Cahill et al., 1996; Demas et al.,
1997) and copulatory actions (Harris and Sachs, 1975;
Dominguez et al., 2001), this region is of particular
interest for the study of the hormonal effects on neurogenesis.
Gonadal steroid hormones may exert influential
effects on cognition, memory, and mood in mammals,
including humans (Vermeulen, 1993; Wang et al.,
1996; Wolf and Kirschbaum, 2002). On the cellular
level, these hormones may play a role in the proliferation (Ormerod and Galea, 2001), survival (Leranth et
al., 2000), and activation (Insel, 1990) of neurons.
Interestingly, estrogen administration does produce an
increase in the number of new cells in the DG of adult
female rats and meadow voles (Tanapat et al., 1999;
Ormerod and Galea, 2001) and in the SVZ of adult
female prairie voles (Smith et al., 2001). In the
present study, we examined the effects of the gonadal
steroid hormone, testosterone, and then its metabolites, estrogen and 5␣-dihydrotestosterone (DHT), on
the addition of new cells in the amygdala of male
meadow voles (Microtus pennsylvanicus). We also
identified the neuronal or glial phenotype of the newly
proliferated cells in the amygdala. Finally, a time
course study was performed to assess whether cells in
the amygdala proliferate locally.
chow and sunflower seeds) and water. Subjects were randomly assigned to experimental groups.
Treatment Groups
In the first experiment, subjects (2–3 months old) were
castrated, followed by a recovery period of 2–3 weeks.
Thereafter, they were implanted with Silastic tubing (10 mm
long, 2 mm i.d., 3.2 mm o.d.) filled with oil vehicle (n ⫽ 7)
or testosterone propionate (TP; n ⫽ 7; 0.1 mg/␮L sesame
oil, ⬇20 ␮L each). In the second experiment, subjects (3–5
months old) were castrated, allowed to recover for 2–3
weeks, and were then divided into four groups that received
implants of Silastic tubing filled with either oil vehicle
(control, n ⫽ 5), TP (n ⫽ 6; 0.1 mg/␮L oil), DHT (n ⫽ 7;
0.1 mg/␮L oil), or estradiol benzoate (EB; n ⫽ 6; ⬇1.5 mg
each). In both experiments, subjects received hormonal or
control treatment until sacrifice, which occurred 72 h after
tubing implantation. In the third experiment, intact male
meadow voles (2– 4 months old) that received no hormonal
treatment were used as subjects to examine the presence of
newly proliferated cells. Subjects were sacrificed at 30 min
or 1, 6, or 24 h following an acute injection of 5-bromo-2⬘deoxyuridine (BrdU; n ⫽ 3 for each time point).
Injections of BrdU
To label proliferating cells, subjects were injected with a
cell proliferation marker, BrdU (Sigma, St. Louis, MO). In
experiments 1 and 2, injections began 48 h following tubing
implantation and continued at 6 h intervals during the next
24 h of treatment (total of four injections per animal).
Injections of BrdU were given intraperitoneally (ip; 50 ␮g/g
body weight) in 0.9% NaCl and 0.007 N NaOH, as described previously (Smith et al., 2001). In experiment 3, one
injection of a higher concentration of BrdU (300 ␮g/g body
weight) was given to maximally label the number of proliferating cells, as indicated in a recent report (Cameron and
McKay, 2001), and the subjects were then sacrificed either
30 min or 1, 6, or 24 h later.
MATERIALS AND METHODS
Brain Perfusion/Fixation
Subjects
Subjects were anesthetized with sodium pentobarbital (0.1
mg/10 g body weight) and perfused through the ascending
aorta using 0.9% saline followed by 4% paraformaldehyde
in 0.1 M phosphate buffer solution (PBS; pH 7.4). Brains
were harvested, postfixed for 2 h in 4% paraformaldehyde,
and then stored in 30% sucrose in PBS. Brains were cut into
40 ␮m coronal or sagittal sections on a microtome, and the
sections were stored in 0.1 M PBS with 1% sodium azide
until processing either for peroxidase BrdU immunostaining
or for triple fluorescence immunolabeling.
Subjects were sexually naive adult male meadow voles (M.
pennsylvanicus) that were offspring of the F3 generation of
a laboratory-breeding colony. The voles were weaned at 21
days of age and housed in same-sex sibling pairs in plastic
cages (29 ⫻ 18 ⫻ 13 cm) that contained cedar chip bedding.
All cages were maintained under 14:10 light/dark photoperiod with lights on at 0700. Temperature was kept at 21
⫾ 1°C. Animals were provided ad libitum with food (rabbit
Gonadal Hormones Alter Neurogenesis
BrdU Immunocytochemistry
Floating brain sections at 120 ␮m intervals were processed
for BrdU immunostaining. Sections were treated with 2 N
HCl for 30 min at 60°C and then with 0.1 M borate buffer
at room temperature for 25 min. After rinsing in 0.1 M PBS,
sections were incubated in 0.3% hydrogen peroxide and
10% methanol in 0.1 M PBS for 15 min; 0.5% Triton X-100
in 0.1 M PBS with 10% normal goat serum (blocking
serum) for 60 min; and rat anti-BrdU monoclonal antibody
(1:1,000; Accurate, Westbury, NY) in blocking serum at
4°C overnight. Sections were then rinsed and incubated in
biotinylated goat antirat IgG (1:200; Jackson ImmunoResearch, West Grove, PA) in blocking serum for 2 h at
room temperature. Thereafter, sections were incubated in
ABC Vector Elite in 0.1 M PBS for 90 min and immunoreactivity was revealed using 3⬘-diaminobenzidine (DAB;
Sigma). To reduce variability in the background and to
standardize the staining, sections from all subjects in each
experiment were processed concurrently for BrdU immunostaining. As previously shown (Fowler et al., 2002), controls included processing brain sections without the primary
antibody and processing brain sections from animals that
did not receive injections of BrdU; in either case, BrdU
immunoreactive staining was not detected.
Triple Fluorescence Immunolabeling
To determine the phenotype of the BrdU-positive (BrdU⫹)
cells, floating sections at 120 ␮m intervals were processed
for BrdU, TuJ1, and NG2 fluorescence triple labeling. TuJ1
is a mouse monoclonal IgG that recognizes a neuron-specific class III ␤-tubulin. This tubulin is considered to be the
earliest marker for cells that have begun to differentiate into
neurons (Alexander et al., 1991; Kameda et al., 1993). NG2
is a polyclonal IgG that recognizes NG2, an integral membrane proteoglycan expressed on glial progenitor cells
(Nishiyama et al., 1995); this proteoglycan has been identified in cells that mature into astrocytes (Fidler et al., 1999;
Dawson et al., 2000), oligodendrocytes (Butt and Berry,
2000; Mallon et al., 2002), and microglia (Chang et al.,
2000; Jones et al., 2002). Both TuJ1 and NG2 have been
used to identify the phenotype of newly proliferated cells in
adult brains (Shihabuddin et al., 2000; Fowler et al., 2002).
To triple label, sections were rinsed, blocked with 10%
normal donkey serum in 0.1% Triton X-100 and 0.1 M PBS,
and incubated in rabbit anti-NG2 (1:150; Chemicon) at 4°C
overnight. On day 2, the sections was rinsed and incubated
in Cy5-conjugated donkey antirabbit IgG (1:100; Jackson
ImmunoResearch) for 2 h. Next, the sections were rinsed,
incubated in 10% normal goat serum in 0.1% Triton-0.1 M
PBS, and then incubated in mouse anti-TuJ1 (1:500; Covance) at 4°C overnight. Following rinsing on day 3, the
sections were incubated in Alexa-488- conjugated goat antimouse IgG (1:400; Molecular Probes) for 2 h. Then, they
were processed for BrdU immunocytochemistry by incubating in normal donkey serum, rat anti-BrdU (1:200; Accurate) in 0.1 M PBS with 0.1% Triton X-100 at 4°C 36 h, and
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Texas Red-conjugated donkey antirat IgG (1:200; Jackson
ImmunoResearch) for 2 h at room temperature. Sections
were rinsed, mounted on slides with slowfade component A
(Molecular Probes), and coverslipped.
Data Quantification and Analysis
All slides were coded to disguise group identity. For peroxidase BrdU immunostaining, BrdU⫹ cells were examined
and quantified bilaterally in the DG, central (CeA), medial
(MeA), and cortical (CorA) nuclei of the amygdala, and
ventromedial nucleus of the hypothalamus (VMH) on coronal sections for all groups in experiment 1 and 2. For
experiment 3, BrdU⫹ cells in the CeA, MeA, CorA, and
VMH were examined on sagittal sections and quantified on
coronal sections.
BrdU⫹ cells were visualized under 40X magnification
using a Zeiss AxioskopII microscope. BrdU⫹ cells were
counted in the hilus, granule, and molecular cell layers of
the DG (corresponding to Plates 29 –32 in Paxinos and
Watson, 1998) for seven to eight sections per animal;
groups did not differ in the number of sections counted. In
the central, medial, and cortical nuclei of the amygdala
(Plates 28 –29 in Paxinos and Watson, 1998) and VMH
(Plates 30 –32 in Paxinos and Watson, 1998) three sections
were counted per animal. Furthermore, the areas of these
brain regions were measured using the NIH IMAGE program. The images were projected to a computer screen and
the boundaries of each region were traced bilaterally. Because no region demonstrated an overall left-right asymmetry, both sides were combined and the area within each
tracing was calculated. In all regions, the sections were
carefully matched between animals. Cell counts and area
measurements were averaged over the number of sections
analyzed for each brain area, and cell density (number of
cells per mm2) was calculated for each subject. Group
differences in the size of the region and the density of
BrdU⫹ cells for each region were analyzed either by a t test
(experiment 1) or by a one-way analysis of variance
(ANOVA), followed by a Student-Newman-Keuls (SNK)
post-hoc test (experiments 2 and 3). Because the BrdUlabeled nuclei were ⬇5 ␮m in diameter and sections at 120
␮m intervals were counted, the possibility of counting split
cells on different sections was minimized to less than 10%,
according to the equation of Abercrombie (Guillery and
Herrup, 1997).
BrdU-, TuJ1-, and NG2-labeled cells were quantified in
the amygdala for experiments 1 and 2. Cells were visualized
under 63X magnification using a Zeiss 510NLO confocal
microscope. At least 170 cells were counted per group from
two sections/animal. Individual cells stained for BrdU/TuJ1,
BrdU/NG2, or BrdU alone were counted. Percentages were
calculated for the individual subject’s number of doublelabeled cells divided by the corresponding subject’s total
number of BrdU⫹ cells and multiplied by 100. Group differences in the percentage of BrdU⫹ cells containing a
neuronal (TuJ1) or glial (NG2) marker were analyzed by a
one-way ANOVA.
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Fowler et al.
Figure 1 Photomicrographs of BrdU⫹ cells following oil (control) or testosterone propionate (TP)
treatment. (A,B) In the amygdala, the control group (A) shows less BrdU⫹ cells than the TP-treated
group (B). Scale bar ⫽ 200 ␮m. CeA, central nucleus; CorA, cortical nucleus; MeA, medial nucleus;
opt, optic tract. (C,D) In the DG, the control group (C) did not differ from the TP-treated group (D).
Scale bar ⫽ 100 ␮m. GrL, granule cell layer; Hil, hilus.
Radioimmunoassays (RIAs)
Prior to sacrifice, blood samples were obtained from the
jugular vein. Blood was drawn with 20 ␮L of 0.2 M
EDTA in the syringe to prevent clotting. Following collection, blood was placed in centrifuge tubes on ice and
was then centrifuged at 3,000 rpm for 25 min. The
resulting plasma was stored at ⫺80°C. Plasma concentrations of testosterone were measured in duplicate by a
Coat-a-Count total testosterone kit and of estradiol in
duplicate by a Coat-a-Count estradiol kit (Diagnostic
Products Corp.). For the testosterone kit, plasma from
castrated male voles was added to the standards to control
for nonspecific competition in male vole plasma and to
prevent nonspecific binding of iodinated testosterone.
Differing volumes of vole plasma were parallel to the
standard curve of testosterone and estradiol, thus verifying the kits for use in voles. For the testosterone and
estradiol assays, the sensitivities were 4 and 8 pg/mL,
respectively, and the intra- and interassay coefficients of
variation were both ⬍5%. Sample values were analyzed
by a t test or ANOVA, followed by a SNK post-hoc test.
immunocytochemistry produced dense nuclear staining of cells in specific regions of the vole brain. In the
amygdala, the TP-treated males had a significantly
higher density of BrdU⫹ cells compared to the oiltreated group (t ⫽ 3.81; p ⬍ 0.05) [Figs. 1(A,B) and
2]. Specifically, the TP-treated group displayed a
higher density of BrdU⫹ cells in the cortical (t
⫽ 3.13; p ⬍ 0.05) and medial (t ⫽ 5.47; p ⬍ 0.001)
nuclei, but not in the central nucleus, of the amygdala
(t ⫽ 0.74; p ⬎ 0.05) [Fig. 2(B)]. Group differences
were not found in the DG (t ⫽ 0.24; p ⬎ 0.05) and
VMH (t ⫽ 0.76; p ⬎ 0.05) [Figs. 1(C,D) and 2(A)].
For the control group, the density of BrdU⫹ cells in
the DG was approximately 5.6-times the cell density
in the amygdala and 6.8-times the cell density in the
VMH. Following the TP-treatment, the density of
BrdU⫹ cells in the DG was approximately 2.4-times
the cell density in the amygdala.
Experiment 2
RESULTS
Experiment 1
The size of each measured region did not differ between the two treatment groups (p ⬎ 0.05). BrdU
In the amygdala, the EB-treated group had a significantly higher density of BrdU⫹ cells than did the
DHT- and oil-treated groups (F ⫽ 7.52; p ⬍ 0.05)
[Fig. 3(A)]. Upon performing a post-hoc contrast test,
the TP-treated group appeared to approach significance in having a higher density of BrdU⫹ cells than
Gonadal Hormones Alter Neurogenesis
261
Figure 2 The effects of testosterone propionate (TP) treatment on the density of BrdU⫹ cells
for each area in male meadow voles. (A) TP treatment significantly increased the density of
BrdU⫹ cells in the amygdala (Amy), but not in the dentate gyrus of the hippocampus (DG) or
ventromedial nucleus of the hypothalamus (VMH). (B) In the subnuclei of the amygdala,
TP-treatment induced an increase in the density of BrdU⫹ cells in the cortical (CorA) and
medial (MeA), but not central (CeA), nuclei. *p ⬍ 0.05; error bars indicate standard error of
the mean.
the oil-treated group (p ⫽ 0.059). For the specific
subnuclei, the TP- and EB-treated groups had a higher
density of BrdU⫹ cells in the cortical (F ⫽ 18.14; p
⬍ 0.001) and medial (F ⫽ 9.68; p ⬍ 0.001) nuclei,
but not in the central nucleus (F ⫽ 3.07; p ⬎ 0.05), of
the amygdala than did the DHT- or oil-treated groups;
the latter two groups did not differ from each other
[Fig. 3(B)]. Group differences in the density of
BrdU⫹ cells were not seen in the DG (F ⫽ 1.93; p
⬎ 0.05) or VMH (F ⫽ 0.63; p ⬎ 0.05) [Fig. 3(A)].
Further, no group differences were found for the area
of each brain region measured (p ⬎ 0.05).
The testosterone and estradiol RIAs confirmed
group assignment. The TP-treated group had a higher
level of plasma testosterone (11.4 ⫾ 1.4 ng/mL) than
did the oil- (0.8 ⫾ 0.1 ng/mL) or DHT-treated groups
Figure 3 The effects of testosterone propionate (TP), estrogen benzoate (EB), or 5␣-dihydrotestosterone (DHT) on the density of BrdU⫹ cells for each area in male meadow voles. (A)
EB treatment induced an increase in the density of BrdU⫹ cells in the amygdala (Amy), but not
the dentate gyrus of the hippocampus (DG) or ventromedial hypothalamus (VMH), as compared
to the control. (B) Within subnuclei of the amygdala, an increase in the density of BrdU⫹ cells
following TP or EB treatment was present in the cortical (CorA) and medial (MeA), but not
central (CeA), nuclei. Letters represent the results of the post-hoc test; nonshared letters
indicate statistical difference at p ⬍ 0.05; and error bars indicate standard error of the mean.
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Fowler et al.
Figure 4 Mean plasma testosterone levels in meadow
voles. Testosterone propionate (TP)-treated males displayed
levels similar to males that had mated with a female (Mated),
both of which were significantly higher than sexually naive
males (Naive). Letters represent the results of the post-hoc test;
nonshared letters indicate statistical difference at p ⬍ 0.05; and
error bars indicate standard error of the mean.
(0.9 ⫾ 0.2 ng/mL) (F ⫽ 74.56; p ⬍ 0.001). An
additional testosterone RIA was then performed to
compare TP-treated values against naturally occurring
testosterone levels. The plasma testosterone levels of
TP-treated males (n ⫽ 5) did not differ from males
sacrificed after mating (n ⫽ 3), and both were higher
than sexually naive males (n ⫽ 3) (F ⫽ 12.06; p
⬍ 0.05) (Fig. 4). The EB-treated group had a higher
level of plasma estradiol (1.0 ⫾ 0.3 ng/mL) than did
the oil-treated group (less than detection limits of 8
pg/mL).
To confirm phenotypes, new cells were labeled
with BrdU and a neuronal (TuJ1) or glial (NG2)
marker (Fig. 5). No differences were found in the
percentage of BrdU⫹ cells colocalized with either
TuJ1 or NG2. Overall, approximately 44.2% of the
cells labeled for BrdU and TuJ1, 34.5% for BrdU and
NG2, and 21.3% for BrdU alone (Table 1). The 21.3%
of BrdU-labeled cells that did not colabel with TuJ1
or NG2 may indicate undifferentiated progenitors,
differentiated cells having not yet begun to express
either of these markers, mature neurons that may not
express TuJ1, or mature glial cells no longer containing NG2. Thus, the actual percentage of new neurons
or glia may be higher depending on the eventual fate
of these cells or the presence of other markers.
Experiment 3
A time course experiment was performed on intact,
untreated males to investigate whether cells prolifer-
ate locally within the amygdala or VMH. On coronal
and sagittal sections, BrdU⫹ cells were visualized in
the amygdala and VMH at 30 min, 1, 6, and 24 h
following an acute injection of BrdU [Fig. 7(A)].
BrdU⫹ cells were then quantified on the coronal sections (Fig. 6). In the amygdala, the 1 and 6 h groups
did not differ from each other; however, both groups
had a density of BrdU⫹ cells that was higher than the
30 min group but lower than the 24 h group (F
⫽ 13.25; p ⬍ 0.05). In the MeA, the 24 h group had
a higher density of cells than did the 30 min group (F
⫽ 4.10; p ⬍ 0.05). Group differences in BrdU cell
density were not found in the CorA (F ⫽ 0.15; p
⬎ 0.05) or CeA (F ⫽ 2.74; p ⬎ 0.05) or in the VMH
(F ⫽ 1.31; p ⬎ 0.05). Prior reports have described a
“stream” of cells migrating into the cerebral cortex
(Gould et al., 1999b); however, upon qualitative analysis, we could not visually identify any observable
migratory stream pattern from the SVZ into the amygdala at these time points. BrdU-labeling was considered specific because labeled cells could not be found
in animals that did not receive BrdU injections (negative control). Further, BrdU/TuJ1/NG2 immunolabeling was also performed to identify the phenotype
of the cells present 30 min following the BrdU injection. Cells colocalized with BrdU/TuJ1 and BrdU/
NG2 were identified within the amygdala at this time
point [Fig. 7(B)].
DISCUSSION
In the present study, we found that TP or EB, but not
DHT, exerted site-specific increases in the density of
new cells in the amygdala of adult male meadow
voles. Plasma testosterone levels in the TP-treated
males were similar to males following mating, indicating that hormonal treatment reached physiological
levels. The majority of the new cells displayed neuronal (⬇42%) or glial (⬇35%) phenotypes. Finally,
new cells were identified in the amygdala and VMH
as early as 30 min following an acute injection of
Table 1 Percentage ⴞ SEM of BrdUⴙ Cells DoubleLabeled with a Neuronal (TuJ1) or Glial (NG2)
Marker in the Amygdala
Treatment
BrdU/TuJ1
BrdU/NG2
BrdU Alone
Control
TP
EB
DHT
Overall
41.0 ⫾ 9.4
32.8 ⫾ 8.9
44.7 ⫾ 7.1
58.3 ⫾ 15.9
44.2%
36.8 ⫾ 12.3
46.4 ⫾ 6.4
33.7 ⫾ 6.5
21.0 ⫾ 7.9
34.5%
22.2 ⫾ 5.1
20.9 ⫾ 6.2
21.6 ⫾ 3.0
20.8 ⫾ 9.1
21.3%
Gonadal Hormones Alter Neurogenesis
Figure 5 Confocal laser microscope images of cells stained for BrdU, TuJ1, and/or NG2 in the
amygdala of male meadow voles. (A–D) Images display staining for (A) BrdU (red), (B) TuJ1
(green), (C) NG2 (blue), and (D) all three markers. BrdU and TuJ1 colocalized cells display a yellow
image (arrow), and a BrdU-only labeled cell displays a red image. Scale bar ⫽ 10 ␮m. (E) Zoomed
image of newborn neuron. Views along the y-z axis (right) and x-z axis (below) demonstrate 3D
colocalization of BrdU and TuJ1. Scale bar ⫽ 5 ␮m. (F–I) Images display staining for (F) BrdU
(red), (G) TuJ1 (green), (H) NG2 (blue), and (I) all three markers. BrdU and NG2 colocalized cells
display a purple image (arrow). Scale bar ⫽ 10 ␮m. (J) Zoomed image of newborn glial cells. Views
along the y-z axis (right) and x-z axis (below) demonstrate 3D colocalization of BrdU and NG2.
Scale bar ⫽ 5 ␮m.
Figure 7 (A) Sagittal section photomicrograph of BrdU⫹ cells in the amygdala (Amy) 30 min
after an acute injection of BrdU. Scale bar ⫽ 100 ␮m. The insert displays a higher magnification
of BrdU-labeling within the amygdala. Scale bar ⫽ 5 ␮m. hpc, hippocampal complex; ic, internal
capsule; LV, lateral ventricle; opt, optic tract. (B–D) Confocal laser microscope images of cells
stained for BrdU, TuJ1, and/or NG2 in the amygdala 30 min after an acute injection of BrdU. Cells
displayed staining for (B) BrdU/TuJ1, (C) BrdU/NG2, and (D) BrdU alone. Scale bar ⫽ 10 ␮m.
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Figure 6 The density of BrdU⫹ cells at 30 min, 1, 6, and 24 h following an acute BrdU injection.
(A) In the amygdala (Amy), the 1 and 6 h groups did not differ from each other; however, both
groups had a density of BrdU⫹ cells that was higher than the 30 min group but lower than the 24 h
group. Significant differences were not found in the ventromedial hypothalamus (VMH). (B) Within
subnuclei of the amygdala, in the medial nucleus (MeA), the 24 h group had a higher density of
BrdU⫹ cells than did the 30 min group, but the 1 and 6 h groups did not differ from any other
groups. Differences were not found in the cortical (CorA) or central (CeA) nuclei. Letters represent
the results of the post-hoc test; nonshared letters indicate statistical difference at p ⬍ 0.05; and error
bars indicate standard error of the mean.
BrdU. Together, these data suggest that gonadal steroid hormones affect the number of newly proliferated
cells in the amygdala, most likely by acting through
an estrogenic mechanism, and these effects may be
exerted on cells locally proliferating within the amygdala.
Gonadal Steroid Hormones Influence
Cell Proliferation
In the mammalian brain, testosterone may exert its
effects directly or via aromatization into estrogen
(Naftolin et al., 1975) or reduction into DHT (Lieberburg and McEwen, 1977). We found that TP or EB
treatment increased the density of new cells in the
amygdala, particularly in the MeA and CorA. In contrast, DHT treatment was ineffective. The similarities
between the TP and EB, but not DHT, effects indicate
that testosterone may act through an estrogen receptor
mechanism to affect the proliferation of new cells in
the amygdala. It is interesting to note that testosterone
and its metabolites also act on certain reproductive
behaviors and neurotransmitters in a similar manner.
For example, in male hamsters, implants of testosterone or estradiol, but not DHT, in the MeA stimulate
sexual activity in castrated males (Wood and Newman, 1995; Wood, 1996), and aromatase inhibition
decreases mating-associated behaviors (Steel and
Hutchison, 1987). In gonadectomized rats, vasopres-
sin mRNA in the MeA and serotonin receptor and
transporter mRNAs in the dorsal raphe nucleus display an increased expression with testosterone or estrogen, but not DHT, treatment (Wang and De Vries,
1995; Fink et al., 1999). However, if DHT is given in
combination with estrogen, it increases the number of
AVP mRNA labeled cells in MeA (Wang and De
Vries, 1995). Because androgen receptor immunoreactivity in the MeA of castrated male rats can be
increased by estrogen treatment (Lynch and Story,
2000), the effects of DHT on the central nervous
system may rely on the presence of estrogen (DeVries
et al., 1994). Therefore, we cannot exclude the possibility that DHT may be able to act synergistically
with estrogen to influence cell proliferation.
Several lines of evidence support this possibility of
local gonadal steroid action within the amygdala.
First, estrogen-receptor containing cells are present in
the amygdala, particularly in the MeA and CorA, in a
variety of rodent species, including rats (Pfaff and
Keiner, 1973; Simerly et al., 1990), hamsters (Wood
et al., 1992), and prairie voles (Hnatczuk et al., 1994).
Second, high levels of aromatase activity are found in
the MeA and CorA of male rats, and castration does
not affect these levels (Roselli et al., 1984; Roselli and
Resko, 1987). After testosterone infusions in castrated
male rats, high levels of estrogen can be found in the
amygdala (Lieberburg and McEwen, 1977). Finally,
testosterone or estrogen treatment has been found to
Gonadal Hormones Alter Neurogenesis
affect the neuroanatomy of the amygdala in rodents,
possibly due to changes in neuronal soma size or
neurogenesis. In female rats, estrogen treatment prior
to postnatal day 30 increases MeA volume (Mizukami
et al., 1983), and in adult, castrated male hamsters,
testosterone administration also increases MeA volume (Cooke et al., 2002). Interestingly, the latter
finding could not be attributed to changes in soma
size, presenting the possibility of increased neurogenesis in response to testosterone for this species.
The underlying mechanisms for the steroid regulation of cell proliferation in adulthood are still unknown. However, because steroid hormones are able
to alter the transcription rates of genes (Simerly,
1990), one may speculate that estrogen can induce or
facilitate progenitor cells to enter the cell cycle. Alternatively, estrogen may act on other cells, which, in
turn, cause local progenitor cells to proliferate. For
instance, astrocytes contain steroid hormone receptors
(Finley and Kritzer, 1999) and have been shown to
induce cell proliferation in vitro (Lim and AlvarezBuylla, 1999; Song et al., 2002). In response to steroid
hormones, astrocytes may secrete neurochemicals
such as BDNF (Ikeda et al., 2001), which has been
shown to enhance cell proliferation and survival in the
adult brain (Pencea et al., 2001). Importantly, in prairie voles, a species closely related to meadow voles,
increased BDNF expression is present in the amygdala following estrogen treatment (Liu et al., 2001).
Thus, if stem or progenitor cells are locally situated
within the amygdala, it is feasible that local astrocytes
function to induce these cells to undergo proliferation.
The TP-treated and mated males had similar levels
of plasma testosterone, both of which were higher
than sexually naive males in the present study or
males captured during the breeding season in a previous experiment (⬇1.8 ng/mL; Galea and McEwen,
1999). These data suggest that mating was accompanied by an increase in plasma testosterone, and the TP
treatment resulted in a level of testosterone similar to
naturally occurring physiological levels following
mating in male meadow voles. Indeed, reports have
indicated that testosterone levels become elevated significantly after ejaculation in the closely related male
prairie voles (increase from ⬇1.4 to 8.8 ng/mL;
Gaines et al., 1985), and a comparable level of testosterone in male meadow voles increases the attractiveness of male odors to females (Ferkin et al., 1994).
A drawback in the present study was that blood samples were only taken at sacrifice, so it was unclear
whether this level of testosterone was consistent
throughout the 72 h of treatment. Therefore, the observed effects on cell proliferation may be due to the
265
initial rising levels of infused TP, maintained infusion
rate, or maximal level of TP released.
New Cells Proliferate Locally within the
Amygdala
In a previous study of rats, the rate of migration for
new cells derived from the SVZa into the olfactory
bulb was calculated as 22.8 ␮m/h (Luskin and Boone,
1994). If we assume the same rate for migration into
the amygdala, we would expect it to take about 13 h
for the cell in Figure 7(A) to migrate from the nearest
lateral ventricle into the amygdala. Certainly, we cannot exclude the possibility that new cells from the
SVZa could migrate into the amygdala much faster,
but this rate would need to be at least 26 times faster
than the rate into the olfactory bulb. Therefore, because BrdU⫹ cells were identified in the amygdala 30
min postinjection, BrdU may only be incorporated
during S-phase of the cell cycle, and a very high rate
of migration would be necessary for migration from a
ventricle, one may infer with high probability that
these cells are dividing locally, rather than dividing
and then migrating from a ventricular region. Furthermore, these data suggest that the differences seen in
experiments 1 and 2 occurred due to the actions of the
hormones on locally proliferating progenitors. However, we are currently unable to draw a definitive
conclusion about the presence of stem cells within the
amygdala. It has been estimated that stem cells proliferate approximately once every 4 weeks (Morshead
et al., 1994), producing progenitor cells that may
continue to progress through subsequent divisions
⬇17 h while migrating (Smith and Luskin, 1998).
Thus, it is possible that stem cells are located in the
better-known proliferative population, such as the
SVZ, and produce progenitors that migrate to the
amygdala and continue to proliferate locally within
this region. On the other hand, we cannot discount the
possibility that stem cells are localized within the
adult amygdala, a notion that is currently debatable.
Some data suggest that stem cells are not localized
outside of the ventricular subependyma, for example,
within the adult DG (Seaberg and van Der Kooy,
2002), whereas others suggest that stem cells are
present in the DG and in other non-neurogenic zones
of the adult brain (Palmer et al., 1997, 1999).
Possible Functional Significance of New
Cells in the Amygdala
The amygdala has been implicated in many reproductive-associated functions and behaviors, such as olfactory/pheromonal processing (Luiten et al., 1985;
266
Fowler et al.
Meredith, 1991), social learning and memory (Kirkpatrick et al., 1994; Demas et al., 1997), copulatory
actions (Harris and Sachs, 1975; Dominguez et al.,
2001), and aggressive behavior (Albert and Walsh,
1984; Wang et al., 1997). In the present study, TP or
EB treatment increased the density of new cells in the
amygdala of adult male meadow voles; importantly,
the TP treatment induced plasma testosterone levels
similar to levels found following mating, indicating
the physiological relevance of the findings. In male
meadow voles, testosterone plays an important role in
a male’s attractiveness to a female (Ferkin et al.,
1994) and preference for a specific female’s odor
(Ferkin, 1992). Periods of reproduction have been
correlated with high levels of testosterone and aggression (Boonstra et al., 1994; Galea and McEwen,
1999), and aggression increases a male’s reproductive
success (Storey and Snow, 1990). Although not
known in meadow voles, direct implants of testosterone into the MeA enhance sexual activity in male
hamsters (Wood and Newman, 1995), and the amygdala is implicated in aggressive and social behaviors
in male prairie voles (Kirkpatrick et al., 1994; Wang
et al., 1997). Therefore, it is possible that new cells in
the amygdala may mature and contribute to one or
more of these reproductive-associated functions
and/or behaviors. Recent findings appear to support
the concept that adult-born neurons mature and function in the brain: in male golden hamsters, new neurons in the olfactory bulb become activated following
exposure to females in estrus (Huang and Bittman,
2002), and in mice, a diminished number of new cells
in the DG is correlated with deficits in learning and
memory abilities (Shors et al., 2001).
CONCLUSION
In male meadow voles, hormonal status influences the
density of new neurons and glia in the MeA and
CorA; this effect was site-specific, as differences were
not seen in the DG and VMH. Because increases were
seen with TP and EB, but not DHT, and because
progenitors appear to be proliferating within the
amygdala, it is hypothesized that the hormonal effects
on new cells are most likely occurring via estrogenic
mechanisms locally within the amygdala. In mammals, including humans, gonadal hormones are involved in the development, differentiation, and protection of the central nervous system (Garcia-Segura
et al., 1994; Romeo et al., 2002), and the amygdala
has been implicated in a variety of cognitive and
behavioral functions (Albert and Walsh, 1984; Cahill
et al., 1996). Therefore, in future studies, it will be
both interesting and essential to determine the functional significance of new cell incorporation in the
adult amygdala.
We are grateful to Dr. Yan Liu, Cheryl Pye, and Jenny
Stowe for their technical assistance and to Dr. Thomas
Curtis, Dr. Yan Liu, Brandon Aragona, and Jenny Stowe for
a critical reading of the manuscript. We also thank Dr.
Theresa Lee of the University of Michigan for providing the
meadow vole colony.
REFERENCES
Albert DJ, Walsh ML. 1984. Neural systems and the inhibitory modulation of agonistic behavior: a comparison of
mammalian species. Neurosci Biobehav Rev 8:5–24.
Alexander JE, Hunt DF, Lee MK, Shabanowitz J, Michel H,
Berlin SC, MacDonald TL, Sundberg RJ, Rebhun LI,
Frankfurter A. 1991. Characterization of posttranslational
modifications in neuron-specific class III beta-tubulin by
mass spectrometry. Proc Natl Acad Sci USA 88:4685–
4689.
Bedard A, Bernier PJ, Vinet J, Levesque M, Goulet S,
Parent A. 2001. Neuroblast migratory stream in the temporal lobe of the adult primate. In: Program No. 248.8,
Abstracts Viewer and Itinerary Planner. Washington, DC:
Society for Neuroscience. CD-ROM.
Boonstra R, Hochachka WM, Pavone L. 1994. Heterozygosity, aggression, and population fluctuations in
meadow voles (Micortus pennsylvanicus). Evolution 48:
1350 –1363.
Brezun JM, Daszuta A. 1999. Depletion in serotonin decreases neurogenesis in the dentate gyrus and the subventricular zone of adult rats. Neuroscience 89:999 –1002.
Brezun JM, Daszuta A. 2000. Serotonin may stimulate
granule cell proliferation in the adult hippocampus, as
observed in rats grafted with foetal raphe neurons. Eur
J Neurosci 12:391–396.
Butt AM, Berry M. 2000. Oligodendrocytes and the control
of myelination in vivo: new insights from the rat anterior
medullary velum. J Neurosci Res 59:477– 488.
Cahill L, Haier RJ, Fallon J, Alkire MT, Tang C, Keator D,
Wu J, McGaugh JL. 1996. Amygdala activity at encoding
correlated with long-term, free recall of emotional information. Proc Natl Acad Sci USA 93:8016 – 8021.
Cameron HA, Gould E. 1994. Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. Neuroscience 61:203–209.
Cameron HA, McKay RD. 2001. Adult neurogenesis produces a large pool of new granule cells in the dentate
gyrus. J Comp Neurol 435:406 – 417.
Chang A, Nishiyama A, Peterson J, Prineas J, Trapp BD.
2000. NG2-positive oligodendrocyte progenitor cells in
adult human brain and multiple sclerosis lesions. J Neurosci 20:6404 – 6412.
Gonadal Hormones Alter Neurogenesis
Cooke BM, Hegstrom CD, Breedlove SM. 2002. Photoperiod-dependent response to androgen in the medial amygdala of the Siberian hamster, Phodopus sungorus. J Biol
Rhythms 17:147–154.
Coolen LM, Wood RI. 1999. Testosterone stimulation of the
medial preoptic area and medial amygdala in the control
of male hamster sexual behavior: redundancy without
amplification. Behav Brain Res 98:143–153.
Dawson MR, Levine JM, Reynolds R. 2000. NG2-expressing cells in the central nervous system: are they oligodendroglial progenitors? J Neurosci Res 61:471– 479.
Demas GE, Williams JM, Nelson RJ. 1997. Amygdala but
not hippocampal lesions impair olfactory memory for
mate in prairie voles (Microtus ochrogaster). Am J
Physiol 273:R1683–1689.
DeVries GJ, Wang Z, Bullock NA, Numan S. 1994. Sex
differences in the effects of testosterone and its metabolites on vasopressin messenger RNA levels in the bed
nucleus of the stria terminalis of rats. J Neurosci 14:
1789 –1794.
Dominguez J, Riolo JV, Xu Z, Hull EM. 2001. Regulation
by the medial amygdala of copulation and medial preoptic dopamine release. J Neurosci 21:349 –355.
Ferkin MH. 1992. Time course of androgenic modulation of
odor preferences and odor cues in male meadow voles,
Microtus pennsylvanicus. Horm Behav 26:512–521.
Ferkin MH, Sorokin ES, Renfroe MW, Johnston RE. 1994.
Attractiveness of male odors to females varies directly
with plasma testosterone concentration in meadow voles.
Physiol Behav 55:347–353.
Fidler PS, Schuette K, Asher RA, Dobbertin A, Thornton
SR, Calle-Patino Y, Muir E, Levine JM, Geller HM,
Rogers JH, et al. 1999. Comparing astrocytic cell lines
that are inhibitory or permissive for axon growth: the
major axon-inhibitory proteoglycan is NG2. J Neurosci
19:8778 – 8788.
Fink G, Sumner B, Rosie R, Wilson H, McQueen J. 1999.
Androgen actions on central serotonin neurotransmission:
relevance for mood, mental state and memory. Behav
Brain Res 105:53– 68.
Finley SK, Kritzer MF. 1999. Immunoreactivity for intracellular androgen receptors in identified subpopulations
of neurons, astrocytes and oligodendrocytes in primate
prefrontal cortex. J Neurobiol 40:446 – 457.
Fowler CD, Liu Y, Ouimet C, Wang Z. 2002. The effects of
social environment on adult neurogenesis in the female
prairie vole. J Neurobiol 51:115–128.
Fuchs E, Flugge G, Ohl F, Lucassen P, Vollmann-Honsdorf
GK, Michaelis T. 2001. Psychosocial stress, glucocorticoids, and structural alterations in the tree shrew hippocampus. Physiol Behav 73:285–291.
Gaines MS, Fugate CL, Johnson ML, Johnson DC, Hisey
JR, Quadagno D. 1985. Manipulation of aggressive behavior in male prairie voles (Microtus ochrogaster) implanted with testosterone in silastic tubing. Can J Zool
63:2525–2528.
Galea LAM, McEwen BS. 1999. Sex and seasonal differ-
267
ences in the rate of cell proliferation in the dentate gyrus
of adult wild meadow voles. Neurosci 89:955–964.
Garcia-Segura LM, Chowen JA, Parducz A, Naftolin F.
1994. Gonadal hormones as promoters of structural synaptic plasticity: cellular mechanisms. Prog Neurobiol 44:
279 –307.
Gould E, Beylin A, Tanapat P, Reeves A, Shors TJ. 1999a.
Learning enhances adult neurogenesis in the hippocampal
formation. Nat Neurosci 2:260 –265.
Gould E, McEwen BS, Tanapat P, Galea LA, Fuchs E.
1997. Neurogenesis in the dentate gyrus of the adult tree
shrew is regulated by psychosocial stress and NMDA
receptor activation. J Neurosci 17:2492–2498.
Gould E, Reeves AJ, Graziano MS, Gross CG. 1999b.
Neurogenesis in the neocortex of adult primates. Science
286:548 –552.
Guillery RW, Herrup K. 1997. Quantification without pontification: choosing a method for counting objects in
sectioned tissues. J Comp Neurol 386:2–7.
Harris VS, Sachs BD. 1975. Copulatory behavior in male
rats following amygdaloid lesions. Brain Res 86:514 –
518.
Hnatczuk OC, Lisciotto CA, DonCarlos LL, Carter CS,
Morrell JI. 1994. Estrogen receptor immunoreactivity in
specific brain areas of the prairie vole (Microtus ochrogaster) is altered by sexual receptivity and genetic sex.
J Neuroendocrinol 6:89 –100.
Huang L, Bittman EL. 2002. Olfactory bulb cells generated
in adult male golden hamsters are specifically activated
by exposure to estrous females. Horm Behav 41:343–
350.
Huang L, DeVries GJ, Bittman EL. 1998. Photoperiod
regulates neuronal bromodeoxyuridine labeling in the
brain of a seasonally breeding mammal. J Neurobiol
36:410 – 420.
Ikeda O, Murakami M, Ino H, Yamazaki M, Nemoto T,
Koda M, Nakayama C, Moriya H. 2001. Acute up-regulation of brain-derived neurotrophic factor expression
resulting from experimentally induced injury in the rat
spinal cord. Acta Neuropathol (Berl) 102:239 –245.
Insel TR. 1990. Regional induction of c-fos-like protein in
rat brain after estradiol administration. Endocrinology
126:1849 –1853.
Jones LL, Yamaguchi Y, Stallcup WB, Tuszynski MH.
2002. NG2 is a major chondroitin sulfate proteoglycan
produced after spinal cord injury and is expressed by
macrophages and oligodendrocyte progenitors. J Neurosci 22:2792–2803.
Kameda Y, Kameya T, Frankfurter A. 1993. Immunohistochemical localization of a neuron-specific ␤˜ tubulin Isotype in the developing chicken ultimobranchial glands.
Brain Res 628:121–127.
Kempermann G, Kuhn HG, Gage FH. 1997. More hippocampal neurons in adult mice living in an enriched
environment. Nature 386:493– 495.
Kirkpatrick B, Carter CS, Newman SW, Insel TR. 1994.
Axon-sparing lesions of the medial nucleus of the amygdala decrease affiliative behaviors in the prairie vole
268
Fowler et al.
(Microtus ochrogaster): behavioral and anatomical specificity. Behav Neurosci 108:501–513.
Leranth C, Roth RH, Elswoth JD, Naftolin F, Horvath TL,
Redmond Jr DE. 2000. Estrogen Is Essential for Maintaining Nigrostriatal Dopamine Neurons in Primates: Implications for Parkinson’s Disease and Memory. J Neurosci 20:8604 – 8609.
Lieberburg I, McEwen BS. 1977. Brain cell nuclear retention of testosterone metabolites, 5alpha-dihydrotestosterone and estradiol-17beta, in adult rats. Endocrinology
100:588 –597.
Lim DA, Alvarez-Buylla A. 1999. Interaction between astrocytes and adult subventricular zone precursors stimulates
neurogenesis. Proc Natl Acad Sci USA 96:7526 –7531.
Liu Y, Fowler CD, Young LJ, Yan Q, Insel TR, Wang ZX.
2001. Expression and estrogen regulation of brain-derived
neurotrophic factor gene and protein in the forebrain of
female prairie voles. J Comp Neurol 433:499 –514.
Luiten PG, Koolhaas JM, de Boer S, Koopmans SJ. 1985. The
cortico-medial amygdala in the central nervous system organization of agonistic behavior. Brain Res 332:283–297.
Luskin MB, Boone MS. 1994. Rate and pattern of migration
of lineally related olfactory bulb interneurons generated
postnatally in the subventricular zone of the rat. Chem
Senses 19:695–714.
Lynch CS, Story AJ. 2000. Dihydrotestosterone and estrogen regulation of rat brain androgen-receptor immunoreactivity. Physiol Behav 69:445– 453.
Mallon BS, Shick HE, Kidd GJ, Macklin WB. 2002. Proteolipid promoter activity distinguishes two populations
of NG2-positive cells throughout neonatal cortical development. J Neurosci 22:876 – 885.
Meredith M. 1991. Sensory processing in the main and
accessory olfactory systems: comparisons and contrasts. J
Steroid Biochem Mol Biol 39:601– 614.
Mizukami S, Nishizuka M, Arai Y. 1983. Sexual difference
in nuclear volume and its ontogeny in the rat amygdala.
Exp Neurol 79:569 –575.
Morshead CM, Reynolds BA, Craig CG, McBurney MW,
Staines WA, Morassutti D, Weiss S, van der Kooy D.
1994. Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 13:1071–1082.
Naftolin F, Ryan KJ, Davies IJ, Reddy VV, Flores F, Petro
Z, Kuhn M, White RJ, Takaoka Y, Wolin L. 1975. The
formation of estrogens by central neuroendocrine tissues.
Recent Prog Horm Res 31:295–319.
Nilsson M, Perfilieva E, Johansson U, Orwar O, Eriksson
PS. 1999. Enriched environment increases neurogenesis
in the adult rat dentate gyrus and improves spatial memory. J Neurobiol 39:569 –578.
Nishiyama A, Lin XH, Stallcup WB. 1995. Generation of
truncated forms of the NG2 proteoglycan by cell surface
proteolysis. Mol Biol Cell 6:1819 –1832.
Ormerod BK, Galea LA. 2001. Reproductive status influences cell proliferation and cell survival in the dentate
gyrus of adult female meadow voles: a possible regulatory role for estradiol. Neuroscience 102:369 –379.
Palmer TD, Markakis EA, Willhoite AR, Safar F, Gage FH.
1999. Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions
of the adult CNS. J Neurosci 19:8487– 8497.
Palmer TD, Takahashi J, Gage FH. 1997. The adult rat
hippocampus contains primordial neural stem cells. Mol
Cell Neurosci 8:389 – 404.
Paxinos G, Watson C. 1998. The rat brain in stereotaxic
coordinates, 4th Edition. San Diego: Academic Press.
Pencea V, Bingaman KD, Wiegand SJ, Luskin MB. 2001.
Infusion of brain-derived neurotrophic factor into the
lateral ventricle of the adult rat leads to new neurons in
the parenchyma of the striatum, septum, thalamus, and
hypothalamus. J Neurosci 21:6706 – 6717.
Pfaff D, Keiner M. 1973. Atlas of estradiol-concentrating
cells in the central nervous system of the female rat.
J Comp Neurol 151:121–158.
Romeo RD, Richardson HN, Sisk CL. 2002. Puberty and
the maturation of the male brain and sexual behavior:
recasting a behavioral potential. Neurosci Biobehav Rev
26:381–391.
Roselli CE, Ellinwood WE, Resko JA. 1984. Regulation of
brain aromatase activity in rats. Endocrinology 114:192–
200.
Roselli CE, Resko JA. 1987. The distribution and regulation
of aromatase activity in the central nervous system. Steroids 50:495–508.
Seaberg RM, van Der Kooy D. 2002. Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted
progenitors. J Neurosci 22:1784 –1793.
Shihabuddin LS, Horner PJ, Ray J, Gage FH. 2000. Adult
Spinal Cord Stem Cells Generate Neurons after Transplantation in the Adult Dentate Gyrus. J Neurosci 20:
8727– 8735.
Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould
E. 2001. Neurogenesis in the adult is involved in the
formation of trace memories. Nature 410:372–376.
Simerly RB. 1990. Hormonal control of neuropeptide gene
expression in sexually dimorphic olfactory pathways.
Trends Neurosci 13:104 –110.
Simerly RB, Chang C, Muramatsu M, Swanson LW. 1990.
Distribution of androgen and estrogen receptor mRNAcontaining cells in the rat brain: an in situ hybridization
study. J Comp Neurol 294:76 –95.
Smith CM, Luskin MB. 1998. Cell cycle length of olfactory
bulb neuronal progenitors in the rostral migratory stream.
Dev Dyn 213:220 –227.
Smith MT, Pencea V, Wang Z, Luskin MB, Insel TR. 2001.
Increased number of BrdU-labeled neurons in the rostral
migratory stream of the estrous prairie vole. Horm Behav
39:11–21.
Song H, Stevens C, Gage F. 2002. Astroglia induce neurogenesis from adult neural stem cells. Nature 417:39 – 44.
Steel E, Hutchison JB. 1987. The aromatase inhibitor, 1,4,6androstatriene-3,17-;dione (ATD), blocks testosteroneinduced olfactory behaviour in the hamster. Physiol Behav 39:141–145.
Gonadal Hormones Alter Neurogenesis
Storey AE, Snow DT. 1990. Postimplantation pregnancy
disruptions in meadow voles: relationship to variation in
male sexual and aggressive behavior. Physiol Behav 47:
19 –25.
Tanapat P, Hastings NB, Reeves AJ, Gould E. 1999. Estrogen stimulates a transient increase in the number of new
neurons in the dentate gyrus of the adult female rat.
J Neurosci 19:5792–5801.
van Praag H, Kempermann G, Gage FH. 1999. Running
increases cell proliferation and neurogenesis in the adult
mouse dentate gyrus. Nat Neurosci 2:266 –270.
Vermeulen A. 1993. The male climacterium. Ann Med
25:531–534.
Wang C, Alexander G, Berman N, Salehian B, Davidson T,
McDonald V, Steiner B, Hull L, Callegari C, Swerdloff
RS. 1996. Testosterone replacement therapy improves
mood in hypogonadal men—a clinical research center
study. J Clin Endocrinol Metab 81:3578 –3583.
Wang Z, De Vries GJ. 1995. Androgen and estrogen effects
on vasopressin messenger RNA expression in the medial
269
amygdaloid nucleus in male and female rats. J Neuroendocrinol 7:827– 831.
Wang Z, Hulihan T, Insel TR. 1997. Sexual and social
experience is associated with different patterns of behavior and neural activation in male prairie voles. Brain Res
767:321–332.
Wolf OT, Kirschbaum C. 2002. Endogenous estradiol and
testosterone levels are associated with cognitive performance in older women and men. Horm Behav 41:259 –266.
Wood RI. 1996. Estradiol, but not dihydrotestosterone, in
the medial amygdala facilitates male hamster sex behavior. Physiol Behav 59:833– 841.
Wood RI, Brabec RK, Swann JM, Newman SW. 1992.
Androgen and estrogen concentrating neurons in chemosensory pathways of the male Syrian hamster brain. Brain
Res 596:89 –98.
Wood RI, Newman SW. 1995. The medial amygdaloid
nucleus and medial preoptic area mediate steroidal control of sexual behavior in the male Syrian hamster. Horm
Behav 29:338 –353.